Crystallographic structure of xanthorhodopsin, the light-driven with a dual

Hartmut Lueckea,b,c,1, Brigitte Schobertb, Jason Stagnoa, Eleonora S. Imashevab, Jennifer M. Wangb, Sergei P. Balashovb,1, and Janos K. Lanyib,c,1

Departments of aMolecular Biology and Biochemistry, and bPhysiology and Biophysics, and cCenter for Biomembrane Systems, University of California, Irvine, CA 92697

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved September 16, 2008 (received for review July 23, 2008) Homologous to and even more to proteorho- found recently in the genome of an abundant coastal ocean dopsin, xanthorhodopsin is a light-driven proton pump that, in methylotroph (11) and earlier in the genomes of Gloebacter addition to , contains a noncovalently bound violaceous, Pyrocystis lunula (12), and others. The proteins in this with a function of a light-harvesting antenna. We determined the clade exhibit significantly less homology to the proteorhodopsins structure of this eubacterial membrane protein–carotenoid com- (11). For example, 137 residues (50%) are identical in plex by X-ray diffraction, to 1.9-Å resolution. Although it contains Gloeobacter and xanthorhodopsin, but only 60 resi- 7 transmembrane helices like bacteriorhodopsin and archaerho- dues (22%) in proteorhodopsin and xanthorhodopsin. Although dopsin, the structure of xanthorhodopsin is considerably different considerable sequence differences separate xanthorhodopsin from the 2 archaeal proteins. The crystallographic model for this from the proteorhodopsins (Fig. 1), its structure, the first for a rhodopsin introduces structural motifs for proton transfer during eubacterial proton pump, is likely to be relevant to other the reaction cycle, particularly for proton release, that are dramat- eubacterial retinal-based pumps. ically different from those in other retinal-based transmembrane pumps. Further, it contains a histidine–aspartate complex for Results and Discussion regulating the pKa of the primary proton acceptor not present in Xanthorhodopsin was crystallized from bicelles (13), with a type archaeal pumps but apparently conserved in eubacterial pumps. In I arrangement of stacked bilayers. The structure was solved to addition to aiding elucidation of a more general proton transfer 1.9-Å resolution (Table 1). The P1 unit cell contains 2 molecules mechanism for light-driven energy transducers, the structure de- of xanthorhodopsin with a head-to-tail arrangement somewhat fines also the geometry of the carotenoid and the retinal. The close similar to 2-dimensional crystal forms of bacteriorhodopsin (14) approach of the 2 polyenes at their ring ends explains why the and (15), as well as 3-dimensional crystals of the efficiency of the excited-state energy transfer is as high as Ϸ45%, D85S bacteriorhodopsin mutant (16), sensory rhodopsin II (17, and the 46° angle between them suggests that the chromophore 18), and Anabaena sensory rhodopsin (19). Considering its location is a compromise between optimal capture of light of all function as an ion transporter in the , xanthor- polarization angles and excited-state energy transfer. hodopsin is unlikely to form such dimers in the original Salini- bacter cells. carotenoid antenna ͉ energy transfer ͉ retinal protein ͉ salinixanthin ͉ X-ray structure Location and Binding Site of Carotenoid Antenna. The C40 carot- enoid lies transverse against the outer surface of helix F at a Ϸ54° ncreased efficiency of light harvesting by antennae, such as angle to the membrane normal, buried at the protein–lipid , is common in photosynthetic membranes, large boundary (Fig. 2A). Its keto-ring is immobilized by residues at I ␤ multiprotein systems that contain tens or hundreds of chro- the extracellular ends of helices E and F and by the -ionone ring mophores. The much simpler retinal-based archaeal proton of the retinal (Fig. 2B) and rotated 82° from the plane of the methyl side chains of its polyene chain and therefore from the pumps bacteriorhodopsin and archaerhodopsin lack antennae; ␲ A light collection and proton translocation are performed by a plane of the extended -system (Fig. 2C). The ring C O is not single protein with a single chromophore. Recently, it was shown hydrogen-bonded. The immobilized and acutely out-of-plane orientation of the keto-ring minimizes participation of its 2 that a light-harvesting antenna can function also in a retinal ␲ protein. Xanthorhodopsin (1), of the eubacterium Salinibacter double bonds in the conjugated -system and explains the ruber (2), contains a single energy-donor carotenoid, salinixan- well-resolved vibronic bands of the carotenoid, the lack of a red-shift of the absorption bands upon binding, and the strong thin (3), and a single acceptor, retinal, in a small (25 kDa) BIOPHYSICS membrane protein. Because energy transfer is from the short- CD bands in the visible region (5). The relatively rigid polyene is wedged in a slot on the outside of helix F, with one side formed lived S2 carotenoid level (4), there must be a short distance and favorable geometry between the 2 to account for by the Leu-194 and Leu-197 side chains and the other by the Ile-205 side chain, and its base by Gly-201. The carotenoid its high (40–50%) efficiency. Close interaction of the 2 chro- A mophores is indicated by dependence of the carotenoid confor- glucoside is hydrogen-bonded to the C O and the NH2 of the mation on the presence of the retinal in the protein (1, 5, 6) and amide side chain of Asn-191, as well as NH1 of Arg-184. The spectral changes of the carotenoid during the photochemical transformations of the retinal (1), but, as for the proteorhodop- Author contributions: S.P.B. and J.K.L. designed research; B.S., E.S.I., J.M.W., S.P.B., and sin family of proteins, no direct structural information has been J.K.L. performed research; E.S.I., J.M.W., and S.P.B. contributed new reagents/analytic tools; available (4, 7). Unexpectedly, the crystallographic structure of H.L., J.S., and J.K.L. analyzed data; and H.L., S.P.B., and J.K.L. wrote the paper. xanthorhodopsin we report here reveals not only the location of The authors declare no conflict of interest. the antenna but also striking differences from the archaeal This article is a PNAS Direct Submission. retinal proteins, bacteriorhodopsin and archaerhodopsin. Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, The photocycle of xanthorhodopsin (8) and the functional www.pdb.org (PDB ID code 3DDL). residues in the ion transfer pathway (1) are similar to those of the 1To whom correspondence may be addressed. E-mail: [email protected], [email protected], or numerous other eubacterial proton pumps, the proteorho- [email protected]. dopsins (9, 10). Proteins homologous to xanthorhodopsin were © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807162105 PNAS ͉ October 28, 2008 ͉ vol. 105 ͉ no. 43 ͉ 16561–16565 Downloaded by guest on September 29, 2021 Fig. 1. Sequence alignment of green light-absorbing proteorhodopsin (PR), xanthorhodopsin (XR), and bacteriorhodopsin (BR), reevaluated from the one shown in ref. 1 by using information gained from the diffraction structure. Red, conserved residues in all three; purple, conserved residues in xanthorho- dopsin and bacteriorhodopsin; yellow, conserved residues in xanthorhodop- sin and proteorhodopsins; blue, residues involved with carotenoid binding. Top row of numbers refer to the xanthorhodopsin sequence; bottom row to the bacteriorhodopsin sequence. Underlining indicates residues in transmem- brane helices. Proteorhodopsin sequence refers to a species from Monterey Bay, MBP1 (protein accession No. AAG10475).

dependence of the carotenoid spectrum on the retinal is ex- plained by the fact that the retinal ␤-ionone ring is part of the carotenoid binding site (Fig. 2B). The keto-ring of the carotenoid is in the space occupied by Trp-138 in bacteriorhodopsin, one of the bulky side chains that confines the retinal ␤-ionone ring in that protein. In xanthor-

Table 1. Data collection and refinement statistics

Data collection Beamline 9.1, SSRL, Menlo Park, CA Wavelength, Å 0.979 Fig. 2. Location of salinixanthin (orange) and retinal (magenta) in xanthor- Space group P1 hodopsin. (A) The extended carotenoid is tightly bound on the transmem- Cell dimensions a ϭ 52.7 Å, b ϭ 59.5 Å, c ϭ 59.7 Å brane surface of xanthorhodopsin, traversing nearly the entire bilayer, with ␣ ϭ 76.4°, ␤ ϭ 74.9°, ␥ ϭ 64.1° an inclination of 54° to the membrane normal. Its keto-ring binds in a pocket Resolution range, Å 45.10–1.90 between helices E and F, very near the ␤-ionone ring of the retinal. The angle Total reflections 166,560 between the chromophore axes is 46°. The angle between the planes of their Unique reflections 46,289 ␲-systems is 68°. Horizontal lines indicate the approximate boundaries of the Redundancy 3.6 (3.5)* lipid bilayer. Helices E and F are marked. (B) The binding pocket of the keto Completeness, % 94.1 (85.5)* ring is formed by Leu-148, Gly-156, Phe-157, Thr-160, Met-208, and Met-211, ␤ Mean I/␴ 8.4 (1.5)* as well as the retinal -ionone ring. (C) The keto-ring of the carotene is rotated 82° out of plane of the salinixanthin- and is in van der Waals Rsym,% 5.7 (48.5)* distance of the retinal ␤-ionone and the phenolic side chain of Tyr-207. Refinement Resolution range, Å 45.10–1.90 No. of reflections used 46,278 hodopsin, it is replaced by a glycine. This residue replacement R /R ,%† 24.7/26.5 work free might be the best diagnostic in a sequence for the possibility of Rmsd bonds, Å 0.012 Rmsd angles, ° 1.35 binding a salinixanthin-like carotenoid. Another difference is No. of atoms/avg. B, Å2 Glu-141 of xanthorhodopsin, which is alanine in bacteriorho- Protein 3,925/44.2 dopsin but a conserved glutamate in proteorhodopsins and Waters 62/53.2 involved in spectral tuning (20), whose carboxyl oxygens are Ϸ4 Retinal 40/33.4 Å from the retinal ␤-ionone ring methyls. An intriguing question Salinixanthin 140/73.0 is whether other retinal proteins might also have an antenna. Of Lipids 250/67.4 the 12 residues in the xanthorhodopsin carotenoid binding site, Ramachandran plot (favored/ 96.3/3.7/0 7 are conserved in Gloeobacter rhodopsin (homologs of Gly-156, allowed/generously allowed), %‡ Thr-160, Asn-191, Leu-197, Ile-205, Tyr-207, and Met-211). *Values in parentheses are for the highest-resolution shell. Thus, it is probable that this protein can bind the C40 carotenoid † Rfree based on a test set size of 4.7%. of Gloeobacter, echinenone, which contains a keto-ring similar to ‡PROCHECK salinixanthin (21).

16562 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807162105 Luecke et al. Downloaded by guest on September 29, 2021 The distance between the centers of the 2 linear chro- mophores is 11.7 Å. The ring moieties are within5Åofone another, and part of the retinal ␤-ionone ring is in van der Waals distance of the carotenoid keto-ring. Both are in contact with the Tyr-207 ring between them (Fig. 2 B and C). This contrasts with the crystal structure of archaerhodopsin (22), a proton pump with a bacterioruberin carotenoid without antenna function (23), where the corresponding center-to-center interchromophore distance is 17 Å, with the closest approach of bacterioruberin to the retinal at 12 Å. In that protein, the carotenoid may have a structural (22) and possibly photoprotective function. The angle between the axes of the chromophores is 46° (Fig. 2A), somewhat less than the 56 Ϯ 3° estimated from the polarization anisotropy of retinal fluorescence (4). The discrep- ancy may originate from the off-axis orientation of the transition moment, as in rhodopsin (24). Energy transfer is optimal when the chromophores are parallel. However, where the 2 spectra overlap, the probability of absorbing incident light of all polar- ization angles is increased when the 2 chromophores are not parallel. This gain would be optimal if the angle between the chromophores were 90°, but that would preclude energy transfer. Thus, the 46° angle in xanthorhodopsin appears to be a com- promise between optimal chromophore interaction and optimal collection of light of all polarizations. In light-harvesting complexes of photosynthetic , it is the large number of antenna molecules with various orientations (25, 26) that ensures the capture of light of different polarizations.

Structure of Xanthorhodopsin Exhibits Large Differences from Bacte- riorhodopsin. The xanthorhodopsin structure extends the archi- Fig. 3. Displacements of the B–C and F–G interhelical segments expose a deep tecture of the growing family of (27). Remarkably, cavity that extends from the extracellular side halfway toward Schiff base. (A) there are greater differences from the main chain of bacte- Comparison of xanthorhodopsin and bacteriorhodopsin. The antiparallel ␤-sheet riorhodopsin than in any of the crystallized microbial rho- of the B–C segment of xanthorhodopsin (gray) packs against the N terminus dopsins, including halorhodopsin, archaerhodopsin, sensory (Bottom Right), whereas in bacteriorhodopsin (magenta) this segment packs rhodopsin II, and Anabena sensory rhodopsin. Helices A and against the F–G loop (Bottom Left). Helices A–E are viewed from the front; F and G are in the back, as marked. (B) The resulting hydrophilic cavity in xanthorho- G are longer by 4 and 9 residues, respectively, and their tilt and dopsin extends from the extracellular surface to Arg-93 and other buried func- rotation, particularly of helix A, are considerably different tional groups. In bacteriorhodopsin, this region is occupied by the protein and (Fig. 3A). The 28 residues that comprise helix B are 4 residues includes the proton release group composed of Glu-194, Glu-204, and 3 ordered shifted in the sequence toward the C terminus (i.e., toward the water molecules, absent in xanthorhodopsin. Buried residues with functional extracellular side). In bacteriorhodopsin, the interhelical B–C roles in transport are shown to illustrate their proximity to the aqueous interface. antiparallel ␤-sheet interacts with the D–E loop, whereas in xanthorhodopsin it reorients dramatically to interact with the Arg-8 peptide CAO near the N terminus, where it forms a mini positively charged (Arg-82 in this protein) side chain 3-stranded ␤-sheet. As a result, the tip of the B–C loop is toward the glutamate pair, upon protonation of Asp-85 (30). displaced, by 30 Å, toward the periphery of the protein (Fig. This is less likely to occur in the xanthorhodopsin photocycle, 3). Unexpected in a heptahelical membrane protein, this because NH1 and NH2 of Arg-93 are both hydrogen-bonded to produces a large cleft that extends far into the interior and the peptide carbonyl of Gln-229 instead of water molecules. brings functional residues, buried in other rhodopsins, near the aqueous interface (Fig. 3B). In bacteriorhodopsin, Wat-402 receives a hydrogen bond from

the protonated retinal Schiff base and donates hydrogen bonds BIOPHYSICS to the 2 anionic residues Asp-85 and Asp-212 (28, 29), and this arrangement is conserved in xanthorhodopsin (Fig. 4). However, the carboxylate of the homolog of Asp-85, Asp-96 in this protein, is severely rotated, and the hydrogen-bonded aqueous network of water molecules in the extracellular region that facilitate proton release in the photocycle (30) is replaced by hydrogen- bonded residues that are likely to be more resistant to rear- rangement than an aqueous network. In bacteriorhodopsin, a pair of glutamate residues, Glu-194 and Glu-204, stabilizes a hydrogen-bonded aqueous network (31), which is the source of the proton released to the extracellular surface after the retinal Schiff base is deprotonated. Sequence alignment (Fig. 1) shows that the eubacterial pumps, proteorhodopsin and xanthorhodop- Fig. 4. Structure of the retinal, the Schiff base counterion, and the extra- sin, contain only 1 of these acidic groups. Further, in xanthor- cellular region. The counterion to the Schiff base is an aspartate–histidine hodopsin, at least, the single glutamate is far removed from complex. The network of water molecules that leads to the extracellular Arg-93 (Ͼ18 Å vs. 7.3 Å in bacteriorhodopsin). In bacteriorho- surface in bacteriorhodopsin is missing, and Arg-93 interacts primarily with dopsin, release of the proton is triggered by the movement of the protein side chains.

Luecke et al. PNAS ͉ October 28, 2008 ͉ vol. 105 ͉ no. 43 ͉ 16563 Downloaded by guest on September 29, 2021 Fig. 6. Cytoplasmic region, with the proton donor Glu-107 and its link via Wat-502 to the retinal region. As in the other microbial rhodopsins, Wat-501 is hydrogen-bonded tightly between the tryptophan just above the retinal (Trp-200) and the main-chain carbonyl of the residue in the ␲-bulge of helix G (Ala-239). Helices B, C, F, and G are marked.

Fig. 5. Protonation states of the aspartate–histidine counterion complex. The pH of the crystallization (5.6), is below the observed (8) spectral transition proton release complex (38, 39) and as in proteorhodopsin (32), between the protonated and deprotonated forms of the Schiff base counte- there is no release of a proton in the xanthorhodopsin photocycle rion (presumably neutral/zwitterionic and anionic). Thus, it seems likely that at the time the Schiff base becomes deprotonated. Instead, the the crystallographic structure contains the neutral/zwitterionic counterion. sequence of proton release to the extracellular side and uptake from the cytoplasmic side is reversed: proton uptake in the cycle An Asp–His Hydrogen-Bonded Pair Is Part of Counterion to Retinal occurs first, evidently by Glu-107 after it has reprotonated the Schiff Base. One of the distinguishing features of eubacterial retinal Schiff base, and the release to the extracellular surface is delayed until the final photocycle step. The rationale for keeping proton pumps is that the pKa of the primary proton acceptor is not as low as 2.5 in bacteriorhodopsin, but near 7 (32, 33). The counterion protonated until the end of the photocycle might be the same as in bacteriorhodopsin for which it was demonstrated origin of the high pKa, which makes these proteins functional as pumps only at alkaline pH (34), has been an unsolved problem. (40) that a neutralized counterion facilitates thermal retinal In xanthorhodopsin, ND1 of His-62 is hydrogen-bonded to OD1 isomerization, an obligatory step in the reaction cycle. of Asp-96. At 2.42/2.55 Å (in the 2 molecules of the asymmetric unit), this is a very short hydrogen bond, such as found at active Hydrogen-Bonded Aqueous Network in the Cytoplasmic sites of numerous proteins (35). Thus, its proton may be shared Needs Less Rearrangement for Proton Transfer. In the cytoplasmic by the imidazole ring and the carboxylate in a single-well, strong region of bacteriorhodopsin, the proton donor is in an anhydrous environment that constitutes the hydrophobic barrier in the hydrogen bond, and the complex, with an expected pK higher a cytoplasmic half of the protein. This, and the fact that OD1 of than the aspartate alone (36), must be regarded as the Schiff base Thr-46 is an acceptor of its proton (28), raises its pK . The counterion. If analogy with bacteriorhodopsin holds, the an- a aspartic acid becomes a proton donor to the Schiff base during ionic, rather than the neutral complex (Fig. 5), is the proton the photocycle only after the Glu–Thr hydrogen bond is broken acceptor of the Schiff base in the photocycle. However, we and this region is hydrated so as to create a hydrogen-bonded cannot exclude the possibility that the neutral form is the proton chain of 4 water molecules to connect the proton donor to its acceptor, because the carboxylate may accommodate another acceptor (41). In xanthorhodopsin, as in the proteorhodopsins, proton to yield a cationic complex. these residues are replaced by a and a serine (Fig. A histidine at this position is highly conserved in the prote- 1), and they are not hydrogen-bonded to one another. The orhodopsins (see Fig. 1 and ref. 37), making it likely that the carboxyl is hydrogen-bonded to Wat-502 that connects to the aspartate–histidine complex is a general characteristic of eu- peptide carbonyl of Lys-240 (Fig. 6). It appears, therefore, that bacterial pumps. It is likely to account not only for the observed in xanthorhodopsin part of the cytoplasmic hydrogen-bonded high pKa of the counterion in these retinal proteins, but also for chain of water molecules between the retinal and the proton the smaller red shift of the chromophore maximum upon the donor is in position already before photoisomerization of the protonation of the counterion in xanthorhodopsin than in bac- retinal. teriorhodopsin (3–5 nm vs. 40 nm) (8) because sharing of the As in the other structurally characterized microbial rho- proton with the histidine would leave a partial charge on the dopsins, the other cytoplasmic water, Wat-501, is hydrogen- aspartate. However, the much smaller red shift in xanthorho- bonded tightly between the tryptophan located immediately on Ϸ dopsin than in proteorhodopsin ( 30 nm) (33) suggests that the cytoplasmic side of the retinal (Trp-200 in xanthorhodopsin) there must be structural differences that influence the delocal- and the main-chain carbonyl of the residue at the apex of the ization of the proton between the carboxylate and the indole ␲-bulge of helix G (Ala-239), a connection that is broken during ring. the photocycle as the movement of the retinal C20 displaces the Once protonated in the photocycle, the His-62–Asp-96 com- side chain of Trp-200 by Ͼ1 Å (30). Wat-501 possesses one of the plex would be a good candidate for the origin of the proton lowest B factors of any atom in the model, and it is noteworthy released to the medium upon deprotonation of the retinal Schiff that its presence appears to induce a deviation from planarity in base, but at neutral pH, at least, such early proton release does the nearby tyrosine side chain. not occur. Our unpublished measurements of transient proton release and uptake with the pH indicator dye pyranine (per- Materials and Methods formed as described in refs. 32 and 38) indicate that unlike in A membrane fraction enriched in xanthorhodopsin was prepared by washing bacteriorhodopsin, but like its mutants that lack the specialized S. ruber cell membranes 4 times with distilled water, followed by washing 3

16564 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807162105 Luecke et al. Downloaded by guest on September 29, 2021 times with 0.01% dodecyl maltoside in 100 mM NaCl and 5 mM N,N-bis(2- 10 cycles of restrained refinement with the program REFMAC (43), 1 molecule of hydroxyethyl)glycine (pH 8). The resulting membrane fraction was resus- the resulting model was used for a second round of molecular replacement, pended in 30 mM phosphate (pH 5.6) and 1 mM sodium azide and concen- yielding much-improved signal-to-noise ratio with Z scores of 7.32 and 7.10 for trated to contain 5 mg/ml xanthorhodopsin (as estimated from absorption at the two rotation functions, respectively. Maps were improved by 2-fold averag- 560 nm). The protein was solubilized in a bicelle-type medium (13) by adding ing with the program DM (44). Electron density for the salinixanthin carotenoid 1 volume of a 16.7% (wt/wt) dimyristoyl phosphatidylcholine in 20% nonyl was readily visible at this stage. The model was improved by iterative cycles of maltoside to 3 volumes of the xanthorhodopsin preparation, vortexing, and model building and refinement with the program COOT (45) and refinement incubating overnight at 4 °C. with the program CNS (46), initially with and later without NCS restraints. The Crystals (Ϸ30 ϫ 30 ϫ 150 ␮m) were grown at 22 °C over 4–5 months in sitting rmsd values between the final model (molecule A/B) and the search models, drops (on Cryschem plates; Hampton Research), containing 10 ␮L of solubilized bacteriorhodopsin and Anabaena sensory rhodopsin, are 1.91/1.88 Å and 2.00/ xanthorhodopsin, 3 ␮L of 3 M sodium phosphate (pH 5.6), and 2 ␮L of 2.5 mM 1.98 Å, respectively. Sequence identities with bacteriorhodopsin and Anabaena sodium azide. The reservoirs contained 1 mL of 2.5 M or 3 M sodium phosphate sensory rhodopsin are 24.6% and 21.5%, respectively. In addition to the carot- (pH 5.6). After 5-min equilibrations with 5%, 10%, and then 15% ethylene glycol, enoid, numerous lipids and lipid fragments have been included in the model. All the crystals were frozen rapidly in liquid nitrogen. Data were collected at 100 K residues fall within the allowed regions of the Ramachandran plot. Refinement on beamline 9.1 at the Stanford Synchrotron Research Laboratory (SSRL) as 360 statistics are listed in Table 1. The coordinate file, 3DDL, has been deposited at the Protein Data Bank. frames with 1° rotations. Data reduction statistics are listed in Table 1. The structure was solved by iterative molecular replacement, by using a composite model that consisted of helices A and B of Anabaena sensory rhodop- ACKNOWLEDGMENTS. We thank T. Poulos and H. Li (University of California, Irvine) for sharing transportation of crystals and beam time and for help with sin [Protein Data Bank (PDB) ID code 1XIO, residues 4–51] and helices C–G of remote data collection. This work was supported by National Institutes of bacteriorhodopsin (PDB ID code 1C3W, residues 81–231) with the program Health Grant GM29498 and Department of Energy Grant DEFG03-86ER13525 PHASER (42). The first rotation function exhibited low signal-to-noise ratio with (to J.K.L.), U.S. Army Research Office Grant W911NF-06-1-0020 (to S.P.B.), and a top Z score of 4.77. The correct solution was peak 5 with a Z score of 4.73. After National Institutes of Health Grant GM56445 (to H.L.).

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